Neural Progenitor Cell Populations

ABSTRACT

This invention provides populations of neural progenitor cells, differentiated neurons, glial cells, and astrocytes. The populations are obtained by culturing stem cell populations (such as embryonic stem cells) in a cocktail of growth conditions that initiates differentiation, and establishes the neural progenitor population. The progenitors can be further differentiated in culture into a variety of different neural phenotypes, including dopaminergic neurons. The differentiated cell populations or the neural progenitors can be generated in large quantities for use in drug screening and the treatment of neurological disorders.

REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Applications60/205,600, filed May 17, 2000; and 60/257,608, filed Dec. 22, 2000. Thepriority applications are hereby incorporated herein by reference intheir entirety.

TECHNICAL FIELD

This invention relates generally to the field of cell biology ofembryonic cells and neural progenitor cells. More specifically, thisinvention relates to the directed differentiation of human pluripotentstem cells to form cells of the neuronal and glial lineages, usingspecial culture conditions and selection techniques.

BACKGROUND

Repairing the central nervous system is one of the frontiers thatmedical science has yet to conquer. Conditions such as Alzheimer'sdisease, Parkinson's disease, epilepsy, Huntington's disease, and strokecan have devastating consequences for those who are afflicted. Traumaticinjury to the head or the spinal chord can instantly propel someone fromthe bounds of everyday life into the ranks of the disabled.

What makes afflictions of the nervous system so difficult to manage isthe irreversibility of the damage often sustained. A central hope forthese conditions is to develop cell populations that can reconstitutethe neural network, and bring the functions of the nervous system backin line.

For this reason, there is a great deal of evolving interest in neuralprogenitor cells. Up until the present time, it was generally thoughtthat multipotent neural progenitor cells commit early in thedifferentiation pathway to either neural restricted cells or glialrestricted cells. These in turn are thought to give rise to matureneurons, or to mature astrocytes and oligodendrocytes. Multipotentneural progenitor cells in the neural crest also differentiate toneurons, smooth muscle, and Schwann cells. It is hypothesized thatvarious lineage-restricted precursor cells renew themselves and residein selected sites of the central nervous system, such as the spinalchord. Cell lineage in the developing neural tube has been reviewed inthe research literature by Kalyani et al. (Biochem. Cell Biol. 6:1051,1998).

Putative multipotent neuroepithelial cells (NEP cells) have beenidentified in the developing spinal cord. Kalyani et al. (Dev. Biol.186:202, 1997) reported NEP cells in the rat. Mujtaba et al. (Dev. Biol.214:113, 1999) reported NEP cells in the mouse. Differentiation of NEPcells is thought to result in formation of restricted precursor cellshaving characteristic surface markers.

Putative neural restricted precursors (NRP) were characterized byMayer-Proschel et al. (Neuron 19:773, 1997). These cells expresscell-surface PS-NCAM, a polysialylated isoform of the neural celladhesion molecule. They reportedly have the capacity to generate varioustypes of neurons, but do not form glial cells.

Putative glial restricted precursors (GRPs) were identified by Rao etal. (Dev. Biol. 188: 48, 1997). These cells apparently have the capacityto form glial cells but not neurons.

Ling et al. (Exp. Neurol. 149:411, 1998) isolated progenitor cells fromthe germinal region of rat fetal mesencephalon. The cells were grown inEGF, and plated on poly-lysine coated plates, whereupon they formedneurons and glia, with occasional tyrosine hydroxylase positive(dopaminergic) cells, enhanced by including IL-1, IL-11, LIF, and GDNFin the culture medium.

Wagner et al. (Nature Biotechnol. 17:653, 1999) reported cells with aventral mesencephalic dopaminergic phenotype induced from animmortalized multipotent neural stem cell line. The cells weretransfected with a Nurr1 expression vector, and then cocultured with VMtype 1 astrocytes. Over 80% of the cells obtained were claimed to have aphenotype resembling endogenous dopaminergic neurons.

Mujtaba et al. (supra) reported isolation of NRP and GRP cells frommouse embryonic stem (mES) cells. The NRPs were PS-NCAM immunoreactive,underwent self-renewal in defined medium, and differentiated intomultiple neuronal phenotypes. They apparently did not form glial cells.The GRPs were A2B5-immunoreactive, and reportedly differentiated intoastrocytes and oligodendrocytes, but not neurons.

A number of recent discoveries have raised expectations that embryoniccells may become a pluripotential source for cells and tissues useful inhuman therapy. Pluripotent cells are believed to have the capacity todifferentiate into essentially all types of cells in the body (R. A.Pedersen, Scientif. Am. 280(4):68, 1999). Early work on embryonic stemcells was done using inbred mouse strains as a model (reviewed inRobertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil.Dev. 6:543, 1994).

Compared with mouse ES cells, monkey and human pluripotent cells haveproven to be much more fragile, and do not respond to the same cultureconditions. Only recently have discoveries been made that allow primateembryonic cells to be cultured ex vivo.

Thomson et al. (Proc. Natl. Acad. Sci. USA 92:7844, 1995) were the firstto successfully culture embryonic stem cells from primates, using rhesusmonkeys and marmosets as a model. They subsequently derived humanembryonic stem (hES) cell lines from human blastocysts (Science 282:114,1998). Gearhart and coworkers derived human embryonic germ (hEG) celllines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad.Sci. USA 95:13726, 1998). Both hES and hEG cells have the long-soughtcharacteristics of human pluripotent stem (hPS) cells: they are capableof ongoing proliferation in vitro without differentiating, they retain anormal karyotype, and they retain the capacity to differentiate toproduce all adult cell types.

Reubinoff et al. (Nature Biotechnol. 18:399, 2000) reported somaticdifferentiation of human blastocysts. The cells differentiatedspontaneously in culture, with no consistent pattern of structuralorganization. After culturing for 4-7 weeks to high density,multicellular aggregates formed above the plane of the monolayer.Different cells in the culture expressed a number of differentphenotypes, including expression of β-actin, desmin, and NCAM.

Spontaneous differentiation of pluripotent stem cells in culture or interatomas generates cell populations with a highly heterogeneous mixtureof phenotypes, representing a spectrum of different cell lineages. Formost therapeutic purposes, it is desirable for differentiated cells tobe relatively uniform—both in terms of the phenotypes they express, andthe types of progeny they can generate.

Accordingly, there is a pressing need for technology to generate morehomogeneous differentiated cell populations from pluripotent cells ofhuman origin.

SUMMARY

This invention provides a system for efficient production of primatecells that have differentiated from pluripotent cells into cells of theneuronal or glial lineage. Populations of cells are described whichcontain precursors for either lineage, which provide a source forgenerating additional precursor cells, the mature cells of the centralnervous system: neurons, astrocytes, or oligodendrocytes. Certainembodiments of the invention have the ability to generate cells of bothlineages. The precursor and mature cells of this invention can be used anumber of important applications, including drug testing and therapy torestore nervous system function.

One embodiment of this invention is a cell population that proliferatesin an in vitro culture, obtained by differentiating primate pluripotentstem (pPS) cells, wherein at least about 30% of the cells in thepopulation are committed to form neuronal cells, glial cells, or both. Asecond embodiment is a cell population that proliferates in an in vitroculture, comprising at least about 60% neural progenitor cells, whereinat least 10% of the cells can differentiate into neuronal cells, and atleast 10% of the cells can differentiate into glial cells. A thirdembodiment is a cell population that proliferates in an in vitroculture, comprising at least about 60% neural progenitor cells, whereinat least 10% of the cells express A2B5, and at least 10% of the cellsexpress NCAM.

Certain cell populations of the invention are obtained bydifferentiating primate pluripotent stem cells, such as human embryonicstem cells. Some are obtained by differentiating stem cells in a mediumcontaining at least two or more ligands that bind growth factorreceptors. Some are obtained by differentiating pPS cells in a mediumcontaining growth factors, sorting the differentiated cells forexpression of NCAM or A2B5, and then collecting the sorted cells.Certain cell populations are enriched such that at least 70% of thecells express NCAM or A2B5.

Another embodiment of this invention is a cell population comprisingmature neurons, astrocytes, oligodendrocytes, or any combinationthereof, obtained by further differentiating a precursor cell populationof this invention. Some such populations are obtained by culturingneural precursors in a medium containing an activator of cAMP, aneurotrophic factor, or a combination of such factors. As describedbelow, neurons produced by such methods may be capable of exhibiting anaction potential, may show gated sodium and potassium channels, and mayshow calcium flux when administered with neurotransmitters or theirequivalents. Included are populations of cells containing a substantialproportion of dopaminergic neurons, detectable for example by stainingfor tyrosine hydroxylase.

Also embodied in the invention are isolated neural precursor cells,neurons, astrocytes, and oligodendrocytes, obtained by selecting a cellfor the desired phenotype from one of the cell populations.

Where derived from an established line of pPS cells, the cellpopulations and isolated cells of this invention will typically have thesame genome as the line from which they are derived. This means that thechromosomal DNA will be over 90% identical between the pPS cells and theneural cells, which can be inferred if the neural cells are obtainedfrom the undifferentiated line through the course of normal mitoticdivision. Neural cells that have been treated by recombinant methods tointroduce a transgene (such as TERT) or knock out an endogenous gene arestill considered to have the same genome as the line from which they arederived, since all non-manipulated genetic elements are preserved.

A further embodiment of the invention is a method of screening acompound for neural cell toxicity or modulation, in which a culture isprepared containing the compound and a neural cell or cell population ofthis invention, and any phenotypic or metabolic change in the cell thatresults from contact with the compound is determined.

Yet another embodiment of the invention is a method for obtaining apolynucleotide comprising a nucleotide sequence contained in an mRNAmore highly expressed in neural progenitor cells or differentiatedcells, as described and exemplified further on in this disclosure. Thenucleotide sequence can in turn be used to produce recombinant orsynthetic polynucleotides, proteins, and antibodies for gene productsenriched or suppressed in neural cells. Antibodies can also be obtainedby using the cells of this invention as an immunogen or an adsorbent toidentify markers enriched or suppressed in neural cells.

A further embodiment of the invention is a method of reconstituting orsupplementing central nervous system (CNS) function in an individual, inwhich the individual is administered with an isolated cell or cellpopulation of this invention. The isolated cells and cell populationscan be used in the preparation of a medicament for use in clinical andveterinary treatment. Medicaments comprising the cells of this inventioncan be formulated for use in such therapeutic applications.

Other embodiments of the invention are methods for obtaining the neuralprecursor cells and fully differentiated cells of this invention, usingthe techniques outlined in this disclosure on a suitable stem cellpopulation. Included are methods for producing cell populationscontaining dopaminergic cells at a frequency of 1%, 3% or 5%—andpopulations of progenitor cells capable of generating dopaminergic cellsat this frequency—from primate embryonic stem cells. This isparticularly significant in view of the loss in dopamine neuron functionthat occurs in Parkinson's disease. The compositions, methods, andtechniques described in this disclosure hold considerable promise foruse in diagnostic, drug screening, and therapeutic applications.

These and other embodiments of the invention will be apparent from thedescription that follows.

DRAWINGS

FIG. 1 is a graph representing the growth of cells bearing neuralmarkers that were derived from human embryonic stem cells. The upperpanel shows growth of cells maintained in the presence of CNTF, bFGF,and NT3, and then sorted for expression of NCAM. The lower panel showsgrowth of cells maintained in the presence of EGF, bFGF, PDGF, andIGF-1, and then sorted for expression of A2B5. Four different hES celllines were used: H1, H7, H9, and H13. The A2B5 selected population hasbeen passaged over 7 times, and can be further differentiated into bothneuronal and glial cells.

FIG. 2 is a schematic diagram outlining an exemplary procedure forobtaining A2B5-positive cells. Abbreviations used: MEF-CM=mediumconditioned by culturing with mouse embryonic fibroblasts; +/−SHH=withor without sonic hedgehog; D/F12=DMEM/F12 medium; N2 and B27, culturesupplements (Gibco); EPFI=differentiation agents EGF, PDGF, bFGF, andIGF-1; PLL=poly-L lysine substrate; PLL/FN=substrate of poly-L lysineand fibronectin.

FIG. 3 is a half-tone reproduction of a fluorescence micrograph of thebrains from neonatal rats administered with cells that express greenfluorescent protein. Left panels: parental hES cell line. Middle panels:embryoid body cells formed from the parental line. Right panels:differentiated cells sorted for expression of NCAM. Undifferentiated hEScells and embryoid body cells remain in the area of administration andshow evidence of necrosis. In contrast, the differentiated NCAM⁺ cellsappear as single cells, and have migrated away from the injection site.

FIG. 4 is a photocopy reproduction of a fluorescence micrograph showinga cell staining for tyrosine hydroxylase (TH), a marker for dopaminergiccells. Embryoid bodies made from human ES cells were maintained in 10 μmretinoic acid for 4 days, plated into a neural-supportive cocktail, andthen passaged into medium containing 10 ng/mL NT-3 and 10 ng/mL BDNF.Certain populations of this invention contain >1% TH-positive cells.

FIGS. 5 A, B and C is a series of graphs showing response of theneural-restricted precursors to various neurotransmitters. FIG. 5A showsthe ratio of emission data from single cells on two differentcoverslips. Both cells responded to GABA, elevated potassium,acetylcholine and ATP. FIG. 5B shows the frequency of cells tested thatresponded to specific neurotransmitters. FIG. 5C shows the combinationsof neurotransmitter responses observed.

FIGS. 6 A, B and C is a series of graphs showing electrophysiology ofneural-restricted precursors. FIG. 6 A shows sodium and potassiumcurrents observed in two cells depolarized to test potentials between−80 and 80 mV from a holding potential of −100 mV. FIG. 6 B shows theinward (Nat) and outward (K) peak current-voltage relationshipsobserved. FIG. 6 C shows action potentials generated by the same cells nresponse to depolarizing stimuli. These measurements show that neuralprecursor cells derived from human ES cells are capable of generatingaction potentials characteristic of neurotransmission.

DETAILED DESCRIPTION

This invention provides a system for preparing and characterizing neuralprogenitor cells, suitable for use for therapeutic administration anddrug screening.

It has been discovered that when pluripotent stem cells are cultured inthe presence of selected differentiating agents, a population of cellsis derived that has a remarkably high proportion of cells withphenotypic characteristics of neural cells. Optionally, the proportionof neural cells can be enhanced by sorting differentiated cellsaccording to cell-surface markers. Since certain types of pluripotentstem cells (such as embryonic stem cells) can proliferate in culture fora year or more, the invention described in this disclosure provides analmost limitless supply of neural precursors. Certain cell populationsof this invention are capable of generating cells of the neuronal orglial lineage, and themselves can be replicated through a large numberof passages in culture.

FIG. 1 shows the growth curve of cells that have been cultured withdifferentiating agents, and then selected according to whether they bearpolysialylated NCAM, or the A2B5 epitope. Either of these cellpopulations can be proliferated through a large number of celldoublings.

Differentiated cells positively selected for A2B5 expression comprisecells that appear to express A2B5 without NCAM, and cells that expressA2B5 and NCAM simultaneously. In one of the experiments described below,maturation of these cells produced 13% oligodendrocytes, and 38%neurons. Since these cells proliferate in long-term culture withoutlosing their phenotype, the population can provide a reserve ofmultipotential cells. Upon administration to a subject with CNSdysfunction, the population would comprise cells that may repopulateboth the neuronal and glial cell lineage, as needed.

If desired, the neural precursor cells can be further differentiated exvivo, either by culturing with a maturation factor, such as aneurotrophic factor, or by withdrawing one or more factors that sustainprecursor cell renewal. Neurons, astrocytes, and oligodendrocytes aremature differentiated cells of the neural lineage that can be obtainedby culturing the precursor cells in this fashion. The neurons obtainedby these methods have extended processes characteristic of this celltype, show staining for neuron-specific markers like neurofilament andMAP-2, and show evidence of synapse formation, as detected by stainingfor synaptophysin. FIG. 5 shows that these cells respond to a variety ofneurotransmitter substances. FIG. 6 shows that these cells are capableof action potentials as measured in a standard patch-clamp system. Inall these respects, the cells are apparently capable of fullneurological function.

Of particular interest is the ability of this system to generate asupply of dopaminergic neurons (FIG. 4). Cells of this type areparticularly desirable for the treatment of Parkinson's disease, forwhich the best current modality is an allograft of fetal brain tissue.The use of fetal tissue as a clinical therapy is fraught with supply andprocedural issues, but no other source described previously can supplythe right kind of cells with sufficient abundance. The neural precursorcells of this invention are capable of generating differentiated cellsin which several percent of the neurons have a dopaminergic phenotype.This is believed to be a sufficient proportion for cell replacementtherapy in Parkinson's disease, and warrants the development of theprogenitor populations of this invention for therapeutic use.

Since pluripotent stem cells and some of the lineage-restrictedprecursors of this invention proliferate extensively in culture, thesystem described in this disclosure provides an unbounded supply ofneuronal and glial cells for use in research, pharmaceuticaldevelopment, and the therapeutic management of CNS abnormalities. Thepreparation and utilization of the cells of this invention isillustrated further in the description that follows.

DEFINITIONS

For the purposes of this disclosure, the terms “neural progenitor cell”or “neural precursor cell” mean a cell that can generate progeny thatare either neuronal cells (such as neuronal precursors or matureneurons) or glial cells (such as glial precursors, mature astrocytes, ormature oligodendrocytes). Typically, the cells express some of thephenotypic markers that are characteristic of the neural lineage.Typically, they do not produce progeny of other embryonic germ layerswhen cultured by themselves in vitro, unless dedifferentiated orreprogrammed in some fashion.

A “neuronal progenitor cell” or “neuronal precursor cell” is a cell thatcan generate progeny that are mature neurons. These cells may or may notalso have the capability to generate glial cells.

A “glial progenitor cell” or “glial precursor cell” is a cell that cangenerate progeny that are mature astrocytes or mature oligodendrocytes.These cells may or may not also have the capability to generate neuronalcells.

A “multipotent neural progenitor cell population” is a cell populationthat has the capability to generate both progeny that are neuronal cells(such as neuronal precursors or mature neurons), and progeny that areglial cells (such as glial precursors, mature astrocytes, or matureoligodendrocytes), and sometimes other types of cells. The term does notrequire that individual cells within the population have the capabilityof forming both types of progeny, although individual cells that aremultipotent neural progenitors may be present.

In the context of cell ontogeny, the adjective “differentiated” is arelative term. A “differentiated cell” is a cell that has progressedfurther down the developmental pathway than the cell it is beingcompared with. Thus, pluripotent embryonic stem cells can differentiateto lineage-restricted precursor cells, such as hematopoetic cells, whichare pluripotent for blood cell types; hepatocyte progenitors, which arepluripotent for hepatocytes; and various types of neural progenitorslisted above. These in turn can be differentiated further to other typesof precursor cells further down the pathway, or to an end-stagedifferentiated cell, which plays a characteristic role in a certaintissue type, and may or may not retain the capacity to proliferatefurther. Neurons, astrocytes, and oligodendrocytes are all examples ofterminally differentiated cells.

A “differentiation agent”, as used in this disclosure, refers to one ofa collection of compounds that are used in culture systems of thisinvention to produce differentiated cells of the neural lineage(including precursor cells and terminally differentiated cells). Nolimitation is intended as to the mode of action of the compound. Forexample, the agent may assist the differentiation process by inducing orassisting a change in phenotype, promoting growth of cells with aparticular phenotype or retarding the growth of others, or acting inconcert with other agents through unknown mechanisms.

Unless explicitly indicated otherwise, the techniques of this inventioncan be brought to bear without restriction on any type of progenitorcell capable of differentiating into neuronal or glial cells.

Prototype “primate Pluripotent Stem cells” (pPS cells) are pluripotentcells derived from pre-embryonic, embryonic, or fetal tissue at any timeafter fertilization, and have the characteristic of being capable underappropriate conditions of producing progeny of several different celltypes that are derivatives of all of the three germinal layers(endoderm, mesoderm, and ectoderm), according to a standard art-acceptedtest, such as the ability to form a teratoma in 8-12 week old SCID mice.

Included in the definition of pPS cells are embryonic cells of varioustypes, exemplified by human embryonic stem (hES) cells, described byThomson et al. (Science 282:1145, 1998); embryonic stem cells from otherprimates, such as Rhesus stem cells (Thomson et al., Proc. Natl. Acad.Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol.Reprod. 55:254, 1996) and human embryonic germ (hEG) cells (Shamblott etal., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Other types ofpluripotent cells are also included in the term. Any cells of primateorigin that are capable of producing progeny that are derivatives of allthree germinal layers are included, regardless of whether they werederived from embryonic tissue, fetal tissue, or other sources. The pPScells are not derived from a malignant source. It is desirable (but notalways necessary) that the cells be karyotypically normal.

pPS cell cultures are described as “undifferentiated” when a substantialproportion of stem cells and their derivatives in the population displaymorphological characteristics of undifferentiated cells, clearlydistinguishing them from differentiated cells of embryo or adult origin.Undifferentiated pPS cells are easily recognized by those skilled in theart, and typically appear in the two dimensions of a microscopic view incolonies of cells with high nuclear/cytoplasmic ratios and prominentnucleoli. It is understood that colonies of undifferentiated cellswithin the population will often be surrounded by neighboring cells thatare differentiated.

“Feeder cells” or “feeders” are terms used to describe cells of one typethat are co-cultured with cells of another type, to provide anenvironment in which the cells of the second type can grow. For example,certain types of pPS cells can be supported by primary mouse embryonicfibroblasts, immortalized mouse embryonic fibroblasts, or humanfibroblast-like cells differentiated from hES cell. pPS cell populationsare said to be “essentially free” of feeder cells if the cells have beengrown through at least one round after splitting in which fresh feedercells are not added to support the growth of the pPS.

The term “embryoid bodies” is a term of art synonymous with “aggregatebodies”. The terms refer to aggregates of differentiated andundifferentiated cells that appear when pPS cells overgrow in monolayercultures, or are maintained in suspension cultures. Embryoid bodies area mixture of different cell types, typically from several germ layers,distinguishable by morphological criteria and cell markers detectable byimmunocytochemistry.

A “growth environment” is an environment in which cells of interest willproliferate, differentiate, or mature in vitro. Features of theenvironment include the medium in which the cells are cultured, anygrowth factors or differentiation-inducing factors that may be present,and a supporting structure (such as a substrate on a solid surface) ifpresent.

A cell is said to be “genetically altered”, “transfected”, or“genetically transformed” when a polynucleotide has been transferredinto the cell by any suitable means of artificial manipulation, or wherethe cell is a progeny of the originally altered cell that has inheritedthe polynucleotide. The polynucleotide will often comprise atranscribable sequence encoding a protein of interest, which enables thecell to express the protein at an elevated level. The genetic alterationis said to be “inheritable” if progeny of the altered cell have the samealteration.

The term “antibody” as used in this disclosure refers to both polyclonaland monoclonal antibody. The ambit of the term deliberately encompassesnot only intact immunoglobulin molecules, but also such fragments andderivatives of immunoglobulin molecules (such as single chain Fvconstructs, diabodies, and fusion constructs) as may be prepared bytechniques known in the art, and retaining a desired antibody bindingspecificity.

General Techniques

For further elaboration of general techniques useful in the practice ofthis invention, the practitioner can refer to standard textbooks andreviews in cell biology, tissue culture, and embryology. Included areTeratocarcinomas and embryonic stem cells: A practical approach (E. J.Robertson, ed., IRL Press Ltd. 1987); Guide to Techniques in MouseDevelopment (P. M. Wasserman et al. eds., Academic Press 1993);Embryonic Stem Cell Differentiation in Vitro (M. V. Wiles, Meth.Enzymol. 225:900, 1993); Properties and uses of Embryonic Stem Cells:Prospects for Application to Human Biology and Gene Therapy (P. D.Rathjen et al., Reprod. Fertil. Dev. 10:31, 1998).

For elaboration of nervous system abnormalities, and thecharacterization of various types of nerve cells, markers, and relatedsoluble factors, the reader is referred to CNS Regeneration: BasicScience and Clinical Advances, M. H. Tuszynski & J. H. Kordower, eds.,Academic Press, 1999.

Methods in molecular genetics and genetic engineering are described inMolecular Cloning: A Laboratory Manual, 2nd Ed. (Sambrook et al., 1989);Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Animal Cell Culture(R. I. Freshney, ed., 1987); the series Methods in Enzymology (AcademicPress); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P.Calos, eds., 1987); Current Protocols in Molecular Biology and ShortProtocols in Molecular Biology, 3rd Edition (F. M. Ausubel et al., eds.,1987 & 1995); and Recombinant DNA Methodology II (R. Wu ed., AcademicPress 1995). Reagents, cloning vectors, and kits for geneticmanipulation referred to in this disclosure are available fromcommercial vendors such as BioRad, Stratagene, Invitrogen, and ClonTech.

General techniques used in raising, purifying and modifying antibodies,and the design and execution of immunoassays includingimmunohistochemistry, the reader is referred to Handbook of ExperimentalImmunology (D. M. Weir & C. C. Blackwell, eds.); Current Protocols inImmunology (J. E. Coligan et al., eds., 1991); and R. Masseyeff, W. H.Albert, and N. A. Staines, eds., Methods of Immunological Analysis(Weinheim: VCH Verlags GmbH, 1993).

Sources of Stem Cells

This invention can be practiced using stem cells of various types, whichmay include the following non-limiting examples.

U.S. Pat. No. 5,851,832 reports multipotent neural stem cells obtainedfrom brain tissue. U.S. Pat. No. 5,766,948 reports producing neuroblastsfrom newborn cerebral hemispheres. U.S. Pat. Nos. 5,654,183 and5,849,553 report the use of mammalian neural crest stem cells. U.S. Pat.No. 6,040,180 reports in vitro generation of differentiated neurons fromcultures of mammalian multipotential CNS stem cells. WO 98/50526 and WO99/01159 report generation and isolation of neuroepithelial stem cells,oligodendrocyte-astrocyte precursors, and lineage-restricted neuronalprecursors. U.S. Pat. No. 5,968,829 reports neural stem cells obtainedfrom embryonic forebrain and cultured with a medium comprising glucose,transferrin, insulin, selenium, progesterone, and several other growthfactors.

Except where otherwise required, the invention can be practiced usingstem cells of any vertebrate species. Included are stem cells fromhumans; as well as non-human primates, domestic animals, livestock, andother non-human mammals.

Amongst the stem cells suitable for use in this invention are primatepluripotent stem (pPS) cells derived from tissue formed after gestation,such as a blastocyst, or fetal or embryonic tissue taken any time duringgestation. Non-limiting examples are primary cultures or establishedlines of embryonic stem cells or embryonic germ cells.

Embryonic Stem Cells

Embryonic stem cells can be isolated from blastocysts of members of theprimate species (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells can be prepared from humanblastocyst cells using the techniques described by Thomson et al. (U.S.Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998) and Reubinoff et al, Nature Biotech. 18:399, 2000.

Briefly, human blastocysts are obtained from human in vivopreimplantation embryos. Alternatively, in vitro fertilized (IVF)embryos can be used, or one-cell human embryos can be expanded to theblastocyst stage (Bongso et al., Hum Reprod 4: 706, 1989). Embryos arecultured to the blastocyst stage in G1.2 and G2.2 medium (Gardner etal., Fertil. Steril. 69:84, 1998). The zona pellucida is removed fromdeveloped blastocysts by brief exposure to pronase (Sigma). The innercell masses are isolated by immunosurgery, in which blastocysts areexposed to a 1:50 dilution of rabbit anti-human spleen cell antiserumfor 30 min, then washed for 5 min three times in DMEM, and exposed to a1:5 dilution of Guinea pig complement (Gibco) for 3 min (Solter et al.,Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two further washes inDMEM, lysed trophectoderm cells are removed from the intact inner cellmass (ICM) by gentle pipetting, and the ICM plated on mEF feeder layers.

After 9 to 15 days, inner cell mass-derived outgrowths are dissociatedinto clumps, either by exposure to calcium and magnesium-freephosphate-buffered saline (PBS) with 1 mM EDTA, by exposure to dispaseor trypsin, or by mechanical dissociation with a micropipette; and thenreplated on mEF in fresh medium. Growing colonies havingundifferentiated morphology are individually selected by micropipette,mechanically dissociated into clumps, and replated. ES-like morphologyis characterized as compact colonies with apparently high nucleus tocytoplasm ratio and prominent nucleoli. Resulting ES cells are thenroutinely split every 1-2 weeks by brief trypsinization, exposure toDulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase(˜200 U/mL; Gibco) or by selection of individual colonies bymicropipette. Clump sizes of about 50 to 100 cells are optimal.

Embryonic Germ Cells

Human Embryonic Germ (hEG) cells can be prepared from primordial germcells present in human fetal material taken about 8-11 weeks after thelast menstrual period. Suitable preparation methods are described inShamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998 and U.S.Pat. No. 6,090,622.

Briefly, genital ridges are rinsed with isotonic buffer, then placedinto 0.1 mL 0.05% trypsin/0.53 mM sodium EDTA solution (BRL) and cutinto <1 mm³ chunks. The tissue is then pipetted through a 100 μL tip tofurther disaggregate the cells. It is incubated at 37° C. for ˜5 min,then ˜3.5 mL EG growth medium is added. EG growth medium is DMEM, 4500mg/L D-glucose, 2200 mg/L mM NaHCO₃; 15% ES qualified fetal calf serum(BRL); 2 mM glutamine (BRL); 1 mM sodium pyruvate (BRL); 1000-2000 U/mLhuman recombinant leukemia inhibitory factor (LIF, Genzyme); 1-2 ng/mlhuman recombinant bFGF (Genzyme); and 10 μM forskolin (in 10% DMSO). Inan alternative approach, EG cells are isolated usinghyaluronidase/collagenase/DNAse. Gonadal anlagen or genital ridges withmesenteries are dissected from fetal material, the genital ridges arerinsed in PBS, then placed in 0.1 ml HCD digestion solution (0.01%hyaluronidase type V, 0.002% DNAse I, 0.1% collagenase type IV, all fromSigma prepared in EG growth medium). Tissue is minced, incubated 1 h orovernight at 37° C., resuspended in 1-3 mL of EG growth medium, andplated onto a feeder layer.

Ninety-six well tissue culture plates are prepared with a sub-confluentlayer of feeder cells (e.g., STO cells, ATCC No. CRL 1503) cultured for3 days in modified EG growth medium free of LIF, bFGF or forskolin,inactivated with 5000 rad γ-irradiation. ˜0.2 mL of primary germ cell(PGC) suspension is added to each of the wells. The first passage isdone after 7-10 days in EG growth medium, transferring each well to onewell of a 24-well culture dish previously prepared with irradiated STOmouse fibroblasts. The cells are cultured with daily replacement ofmedium until cell morphology consistent with EG cells is observed,typically after 7-30 days or 1-4 passages.

Propagation of pPS Cells in an Undifferentiated State

pPS cells can be propagated continuously in culture, using cultureconditions that promote proliferation without promoting differentiation.Exemplary serum-containing ES medium is made with 80% DMEM (such asKnock-Out DMEM, Gibco), 20% of either defined fetal bovine serum (FBS,Hyclone) or serum replacement (WO 98/30679), 1% non-essential aminoacids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Just before use,human bFGF is added to 4 ng/mL (WO 99/20741, Geron Corp.).

Traditionally, ES cells are cultured on a layer of feeder cells,typically fibroblasts derived from embryonic or fetal tissue. Embryosare harvested from a CF1 mouse at 13 days of pregnancy, transferred to 2mL trypsin/EDTA, finely minced, and incubated 5 min at 37° C. 10% FBS isadded, debris is allowed to settle, and the cells are propagated in 90%DMEM, 10% FBS, and 2 mM glutamine. To prepare a feeder cell layer, cellsare irradiated to inhibit proliferation but permit synthesis of factorsthat support ES cells (˜4000 rads γ-irradiation). Culture plates arecoated with 0.5% gelatin overnight, plated with 375,000 irradiated mEFsper well, and used 5 h to 4 days after plating. The medium is replacedwith fresh hES medium just before seeding pPS cells.

Scientists at Geron have discovered that pPS cells can alternatively bemaintained in an undifferentiated state even without feeder cells. Theenvironment for feeder-free cultures includes a suitable culturesubstrate, particularly an extracellular matrix such as Matrigel® orlaminin. The pPS cells are plated at >15,000 cells cm⁻² (optimally90,000 cm⁻² to 170,000 cm⁻²). Typically, enzymatic digestion is haltedbefore cells become completely dispersed (say, ˜5 min with collagenaseIV). Clumps of ˜10-2000 cells are then plated directly onto thesubstrate without further dispersal.

Feeder-free cultures are supported by a nutrient medium typicallyconditioned by culturing irradiated primary mouse embryonic fibroblasts,telomerized mouse fibroblasts, or fibroblast-like cells derived from pPScells. Medium can be conditioned by plating the feeders at a density of˜5-6×10⁴ cm⁻² in a serum free medium such as KO DMEM supplemented with20% serum replacement and 4 ng/mL bFGF. Medium that has been conditionedfor 1-2 days is supplemented with further bFGF, and used to support pPScell culture for 1-2 days.

Under the microscope, ES cells appear with high nuclear/cytoplasmicratios, prominent nucleoli, and compact colony formation with poorlydiscernable cell junctions. Primate ES cells express stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Mouse ES cells can be used as a positive control forSSEA-1, and as a negative control for SSEA-4, Tra-1-60, and Tra-1-81.SSEA-4 is consistently present on human embryonal carcinoma (hEC) cells.Differentiation of pPS cells in vitro results in the loss of SSEA-4,Tra-1-60, and Tra-1-81 expression and increased expression of SSEA-1.SSEA-1 is also found on hEG cells.

Materials and Procedures for Preparing Neural Precursors and TerminallyDifferentiated Cells

Certain neural precursor cells of this invention are obtained byculturing, differentiating, or reprogramming stem cells in a specialgrowth environment that enriches for cells with the desired phenotype(either by outgrowth of the desired cells, or by inhibition or killingof other cell types). These methods are applicable to many types of stemcells, including primate pluripotent stem (pPS) cells described in theprevious section.

Typically, the differentiation takes place in a culture environmentcomprising a suitable substrate, and a nutrient medium to which thedifferentiation agents are added. Suitable substrates include solidsurfaces coated with a positive charge, such as a basic amino acid,exemplified by poly-L-lysine and polyornithine. Substrates can be coatedwith extracellular matrix components, exemplified by fibronectin. Otherpermissive extracellular matrixes include Matrigel® (extracellularmatrix from Engelbreth-Holm-Swarm tumor cells) and laminin. Alsosuitable are combination substrates, such as poly-L-lysine combined withfibronectin, laminin, or both.

Suitable differentiation agents include growth factors of various kinds,such as epidermal growth factor (EGF), transforming growth factor α(TGF-α), any type of fibroblast growth factor (exemplified by FGF-4,FGF-8, and basic fibroblast growth factor=bFGF), platelet-derived growthfactor (PDGF), insulin-like growth factor (IGF-1 and others), highconcentrations of insulin, sonic hedgehog, members of the neurotrophinfamily (such as nerve growth factor=NGF, neurotrophin 3=NT-3,brain-derived neurotrophic factor=BDNF), bone morphogenic proteins(especially BMP-2 & BMP-4), retinoic acid (RA) and ligands to receptorsthat complex with gp130 (such as LIF, CNTF, and IL-6). Also suitable arealternative ligands and antibodies that bind to the respectivecell-surface receptors for the aforementioned factors. Typically, aplurality of differentiation agents is used, which may comprise 2, 3, 4,or more of the agents listed above or in the examples below. Exemplaryis a cocktail containing EGF, bFGF, PDGF, and IGF-1 (Examples 1 and 2).

The factors are supplied to the cells in a nutrient medium, which is anymedium that supports the proliferation or survival of the desired celltype. It is often desirable to use a defined medium that suppliesnutrients as free amino acids rather than serum. It is also beneficialto supplement the medium with additives developed for sustained culturesof neural cells. Exemplary are N2 and B27 additives, availablecommercially from Gibco.

Where the stem cells are pPS cells, the cells (obtained from feeder cellsupported or feeder-free cultures) are differentiated by culturing inthe presence of a suitable cocktail of differentiation agents.

In one method of affecting differentiation, pPS cells are plateddirectly onto a suitable substrate, such as an adherent glass or plasticsurface, such as coverslips coated with a polyamine. The cells are thencultured in a suitable nutrient medium that is adapted to promotedifferentiation towards the desired cell lineage. This is referred to asthe “direct differentiation” method.

In another method, pPS cells are first let differentiate into aheterogeneous cell population. In an exemplary variation, embryoidbodies are formed from the pPS cells by culturing them in suspension.Optionally, one or more of the differentiation agents listed earlier(such as retinoic acid) can be included in the medium to promotedifferentiation within the embryoid body. After the embryoid bodies havereached sufficient size (typically 3-4 days), they are plated onto thesubstrate of the differentiation culture. The embryoid bodies can beplated directly onto the substrate without dispersing the cells. Thisallows neural cell precursors to migrate out of the embryoid bodies andon to the extracellular matrix. Subsequent passaging of these culturesinto an appropriate medium helps select out the neural progenitor cells.

Cells prepared according to these procedures have been found to becapable of further proliferation (Example 1). As many as 30%, 50%, 75%or more of the cells express either polysialylated NCAM or the A2B5epitope, or both. Typically, at least about 10%, 20%, 30% or 50% of thecells express NCAM, and at least about 10%, 20%, 30% or 50% of the cellsexpress A2B5—which implies that they have the capacity to form cells ofthe neuronal lineage, and the glial lineage, respectively.

Optionally, the differentiated cells can be sorted based on phenotypicfeatures to enrich for certain populations. Typically, this will involvecontacting each cell with an antibody or ligand that binds to a markercharacteristic of neural cells, followed by separation of thespecifically recognized cells from other cells in the population. Onemethod is immunopanning, in which specific antibody is coupled to asolid surface. The cells are contacted with the surface, and cells notexpressing the marker are washed away. The bound cells are thenrecovered by more vigorous elution. Variations of this are affinitychromatography and antibody-mediated magnetic cell sorting. In a typicalsorting procedure, the cells are contacted with a specific primaryantibody, and then captured with a secondary anti-immunoglobulin reagentbound to a magnetic bead. The adherent cells are then recovered bycollecting the beads in a magnetic field.

Another method is fluorescence-activated cell sorting, in which cellsexpressing the marker are labeled with a specific antibody, typically byway of a fluorescently labeled secondary anti-immunoglobulin. The cellsare then separated individually according to the amount of bound labelusing a suitable sorting device. Any of these methods permit recovery ofa positively selected population of cells that bear the marker ofinterest, and a negatively selected population of cells that not bearthe marker in sufficient density or accessibility to be positivelyselected. Negative selection can also be effected by incubating thecells successively with a specific antibody, and a preparation ofcomplement that will lyse cells to which the antibody has bound. Sortingof the differentiated cell population can occur at any time, but it hasgenerally been found that sorting is best effected shortly afterinitiating the differentiation process.

It has been found that cells selected positively for polysialylated NCAMcan provide a population that is 60%, 70%, 80%, or even 90% NCAMpositive (Example 1). This implies that they are capable of forming sometype of neural cell, including neurons.

It has also been found that cells selected positively for A2B5 canprovide a population that is 60%, 70%, 80%, or even 90% A2B5 positive(Example 2). This implies that they are capable of forming some type ofneural cell, possibly including both neurons and glial cells. The A2B5positive cells can be sorted again into two separate populations: onethat is A2B5 positive and NCAM negative, and one that is both A2B5positive and NCAM positive.

Differentiated or separated cells prepared according to this procedurecan be maintained or proliferated further in any suitable culturemedium. Typically, the medium will contain most of the ingredients usedinitially to differentiate the cells.

If desired, neural precursor cells prepared according to theseprocedures can be further differentiated to mature neurons, astrocytes,or oligodendrocytes. This can be effected by culturing the cells in amaturation factor, such as forskolin or other compound that elevatesintracellular cAMP levels, such as cholera toxin,isobutylmethylxanthine, dibutyladenosine cyclic monophosphate, or otherfactors such as c-kit ligand, retinoic acid, or neurotrophins.Particularly effective are neurotrophin-3 (NT-3) and brain-derivedneurotrophic factor (BDNF). Other candidates are GDNF, BMP-2, and BMP-4.Alternatively or in addition, maturation can be enhanced by withdrawingsome or all of the factors that promote neural precursor proliferation,such as EGF or FGF.

For use in therapeutic and other applications, it is often desirablethat populations of precursor or mature neurological cells besubstantially free of undifferentiated pPS cells. One way of depletingundifferentiated stem cells from the population is to transfect themwith a vector in which an effector gene under control of a promoter thatcauses preferential expression in undifferentiated cells. Suitablepromoters include the TERT promoter and the OCT-4 promoter. The effectorgene may be directly lytic to the cell (encoding, for example, a toxinor a mediator of apoptosis). Alternatively, the effector gene may renderthe cell susceptible to toxic effects of an external agent, such as anantibody or a prodrug. Exemplary is a herpes simplex thymidine kinase(tk) gene, which causes cells in which it is expressed to be susceptibleto ganciclovir. Suitable pTERT-tk constructs are provided inInternational Patent Publication WO 98/14593 (Morin et al.).

Characteristics of Neural Precursors and Terminally Differentiated Cells

Cells can be characterized according to a number of phenotypic criteria.The criteria include but are not limited to microscopic observation ofmorphological features, detection or quantitation of expressed cellmarkers, enzymatic activity, or neurotransmitters and their receptors,and electrophysiological function.

Certain cells embodied in this invention have morphological featurescharacteristic of neuronal cells or glial cells. The features arereadily appreciated by those skilled in evaluating the presence of suchcells. For example, characteristic of neurons are small cell bodies, andmultiple processes reminiscent of axons and dendrites. Cells of thisinvention can also be characterized according to whether they expressphenotypic markers characteristic of neural cells of various kinds.

Markers of interest include but are not limited to β-tubulin III,microtubule-associated protein 2 (MAP-2), or neurofilament,characteristic of neurons; glial fibrillary acidic protein (GFAP),present in astrocytes; galactocerebroside (GalC) or myelin basic protein(MBP), characteristic of oligodendrocytes; Oct-4, characteristic ofundifferentiated hES cells; Nestin, characteristic of neural precursorsand other cells; and both A2B5 and polysialylated NCAM, as alreadydescribed. While A2B5 and NCAM are instructive markers when studyingneural lineage cells, it should be appreciated that these markers cansometimes be displayed on other cell types, such as liver or musclecells. β-Tubulin III was previously thought to be specific for neuralcells, but it has been discovered that a subpopulation of hES cells isalso β-tubulin III positive. MAP-2 is a more stringent marker for fullydifferentiated neurons of various types.

Tissue-specific markers listed in this disclosure and known in the artcan be detected using any suitable immunological technique—such as flowimmunocytochemistry for cell-surface markers, immunohistochemistry (forexample, of fixed cells or tissue sections) for intracellular orcell-surface markers, Western blot analysis of cellular extracts, andenzyme-linked immunoassay, for cellular extracts or products secretedinto the medium. Expression of an antigen by a cell is said to be“antibody-detectable” if a significantly detectable amount of antibodywill bind to the antigen in a standard immunocytochemistry or flowcytometry assay, optionally after fixation of the cells, and optionallyusing a labeled secondary antibody or other conjugate (such as abiotin-avidin conjugate) to amplify labeling.

The expression of tissue-specific gene products can also be detected atthe mRNA level by Northern blot analysis, dot-blot hybridizationanalysis, or by reverse transcriptase initiated polymerase chainreaction (RT-PCR) using sequence-specific primers in standardamplification methods. See U.S. Pat. No. 5,843,780 for further details.Sequence data for the particular markers listed in this disclosure canbe obtained from public databases such as GenBank (URLwww.ncbi.nlm.nih.gov:80/entrez). Expression at the mRNA level is said tobe “detectable” according to one of the assays described in thisdisclosure if the performance of the assay on cell samples according tostandard procedures in a typical controlled experiment results inclearly discernable hybridization or amplification product. Expressionof tissue-specific markers as detected at the protein or mRNA level isconsidered positive if the level is at least 2-fold, and preferably morethan 10- or 50-fold above that of a control cell, such as anundifferentiated pPS cell, a fibroblast, or other unrelated cell type.

Also characteristic of neural cells, particularly terminallydifferentiated cells, are receptors and enzymes involved in thebiosynthesis, release, and reuptake of neurotransmitters, and ionchannels involved in the depolarization and repolarization events thatrelate to synaptic transmission. Evidence of synapse formation can beobtained by staining for synaptophysin. Evidence for receptivity tocertain neurotransmitters can be obtained by detecting receptors forγ-amino butyric acid (GABA), glutamate, dopamine,3,4-dihydroxyphenylalanine (DOPA), noradrenaline, acetylcholine, andserotonin.

Differentiation of particular neural precursor cell populations of thisinvention (for example, using NT-3 and BDNF) can generate cellpopulations that are at least 20%, 30%, or 40% MAP-2 positive. Asubstantial proportion, say 5%, 10%, 25%, or more of the NCAM or MAP-2positive cells will be capable of synthesizing a neurotransmitter, suchas acetylcholine, glycine, glutamate, norepinephrine, serotonin, orGABA.

Certain populations of the invention contain NCAM or MAP-2 positivecells that are 0.1%, and possibly 1%, 3%, or 5% or more (on a cell countbasis) that are positive for tyrosine hydroxylase (TH), measured byimmunocytochemistry or mRNA expression. This generally considered in theart to be a marker for dopamine synthesizing cells.

To elucidate further mature neurons present in a differentiatedpopulation, the cells can be tested according to functional criteria.For example, calcium flux can be measured by any standard technique, inresponse to a neurotransmitter, or other environmental condition knownto affect neurons in vivo. First, neuron-like cells in the populationare identified by morphological criteria, or by a marker such as NCAM.The neurotransmitter or condition is then applied to the cell, and theresponse is monitored (Example 6). The cells can also be subjected tostandard patch-clamp techniques, to determine whether there is evidencefor an action potential, and what the lag time is between appliedpotential and response. Differentiation of neural precursor populationsof this invention can generate cultures that contain subpopulations thathave morphological characteristics of neurons, are NCAM or MAP-2positive, and show responses with the following frequency: a response toGABA, acetylcholine, ATP, and high sodium concentration in at leastabout 40%, 60% or 80% of the cells; a response to glutamate, glycine,ascorbic acid, dopamine, or norepinephrine in at least about 5%, 10% or20% of the cells. A substantial proportion of the NCAM or MAP-2 positivecells (at least about 25%, 50%, or 75%) can also show evidence of anaction potential in a patch-clamp system.

Other desirable features consistent with functioning neurons,oligodendrocytes, astrocytes, and their precursors can also be performedaccording to standard methods to confirm the quality of a cellpopulation according to this invention, and optimize conditions forproliferation and differentiation of the cells.

Telomerization of Neural Precursors

It is desirable that neural precursors have the ability to replicate incertain drug screening and therapeutic applications, and to provide areservoir for the generation of differentiated neuronal and glial cells.The cells of this invention can optionally be telomerized to increasetheir replication potential, either before or after they progress torestricted developmental lineage cells or terminally differentiatedcells. pPS cells that are telomerized may be taken down thedifferentiation pathway described earlier; or differentiated cells canbe telomerized directly.

Cells are telomerized by genetically altering them by transfection ortransduction with a suitable vector, homologous recombination, or otherappropriate technique, so that they express the telomerase catalyticcomponent (TERT), typically under a heterologous promoter that increasestelomerase expression beyond what occurs under the endogenous promoter.Particularly suitable is the catalytic component of human telomerase(hTERT), provided in International Patent Application WO 98/14592. Forcertain applications, species homologs like mouse TERT (WO 99/27113) canalso be used. Transfection and expression of telomerase in human cellsis described in Bodnar et al., Science 279:349, 1998 and Jiang et al.,Nat. Genet. 21:111, 1999. In another example, hTERT clones (WO 98/14592)are used as a source of hTERT encoding sequence, and spliced into anEcoRI site of a PBBS212 vector under control of the MPSV promoter, orinto the EcoRI site of commercially available pBABE retrovirus vector,under control of the LTR promoter.

Differentiated or undifferentiated pPS cells are genetically alteredusing vector containing supernatants over a 8-16 h period, and thenexchanged into growth medium for 1-2 days. Genetically altered cells areselected using 0.5-2.5 μg/mL puromycin, and recultured. They can then beassessed for hTERT expression by RT-PCR, telomerase activity (TRAPassay), immunocytochemical staining for hTERT, or replicative capacity.The following assay kits are available commercially for researchpurposes: TRAPeze® XL Telomerase Detection Kit (Cat. s7707; IntergenCo., Purchase N.Y.); and TeloTAGGG Telomerase PCR ELISAplus (Cat.2,013,89; Roche Diagnostics, Indianapolis Ind.). TERT expression canalso be evaluated at the mRNA by RT-PCR. Available commercially forresearch purposes is the LightCycler TeloTAGGG hTERT quantification kit(Cat. 3,012,344; Roche Diagnostics). Continuously replicating colonieswill be enriched by further culturing under conditions that supportproliferation, and cells with desirable phenotypes can optionally becloned by limiting dilution.

In certain embodiments of this invention, pPS cells are differentiatedinto multipotent or committed neural precursors, and then geneticallyaltered to express TERT. In other embodiments of this invention, pPScells are genetically altered to express TERT, and then differentiatedinto neural precursors or terminally differentiated cells. Successfulmodification to increase TERT expression can be determined by TRAPassay, or by determining whether the replicative capacity of the cellshas improved.

Other methods of immortalizing cells are also contemplated, such astransforming the cells with DNA encoding myc, the SV40 large T antigen,or MOT-2 (U.S. Pat. No. 5,869,243, International Patent Applications WO97/32972 and WO 01/23555). Transfection with oncogenes or oncovirusproducts is less suitable when the cells are to be used for therapeuticpurposes. Telomerized cells are of particular interest in applicationsof this invention where it is advantageous to have cells that canproliferate and maintain their karyotype—for example, in pharmaceuticalscreening, and in therapeutic protocols where differentiated cells areadministered to an individual in order to augment CNS function.

Use of Neural Precursors and Terminally Differentiated Cells

This invention provides a method to produce large numbers of neuralprecursor cells and mature neuronal and glial cells. These cellpopulations can be used for a number of important research, development,and commercial purposes.

The cells of this invention can be used to prepare a cDNA libraryrelatively uncontaminated with cDNA preferentially expressed in cellsfrom other lineages. For example, multipotent neural progenitor cellsare collected by centrifugation at 1000 rpm for 5 min, and then mRNA isprepared from the pellet by standard techniques (Sambrook et al.,supra). After reverse transcribing into cDNA, the preparation can besubtracted with cDNA from any or all of the following cell types: cellscommitted to the neuronal or glial cell lineage, mature neurons,astrocytes, oligodendrocytes, or other cells of undesired specificity.This produces a select cDNA library, reflecting transcripts that arepreferentially expressed in neuronal precursors compared with terminallydifferentiated cells. In a similar fashion, cDNA libraries can be madethat represent transcripts preferentially expressed in neuronal or glialprecursors, or mature neurons, astrocytes, and oligodendrocytes.

The differentiated cells of this invention can also be used to prepareantibodies that are specific for markers of multipotent neuralprogenitors, cells committed to the neuronal or glial cell lineage, andmature neurons, astrocytes, and oligodendrocytes. This inventionprovides an improved way of raising such antibodies because cellpopulations are enriched for particular cell types compared with pPScell cultures, and neuronal or glial cell cultures extracted directlyfrom CNS tissue.

Polyclonal antibodies can be prepared by injecting a vertebrate animalwith cells of this invention in an immunogenic form. Production ofmonoclonal antibodies is described in such standard references as Harrow& Lane (1988), U.S. Pat. Nos. 4,491,632, 4,472,500 and 4,444,887, andMethods in Enzymology 73B:3 (1981). Other methods of obtaining specificantibody molecules (optimally in the form of single-chain variableregions) involve contacting a library of immunocompetent cells or viralparticles with the target antigen, and growing out positively selectedclones. See Marks et al., New Eng. J. Med. 335:730, 1996, InternationalPatent Applications WO 94/13804, WO 92/01047, WO 90/02809, and McGuinesset al., Nature Biotechnol. 14:1449, 1996. By positively selecting usingpPS of this invention, and negatively selecting using cells bearing morebroadly distributed antigens (such as differentiated embryonic cells) oradult-derived stem cells, the desired specificity can be obtained. Theantibodies in turn can be used to identify or rescue neural cells of adesired phenotype from a mixed cell population, for purposes such ascostaining during immunodiagnosis using tissue samples, and isolatingprecursor cells from terminally differentiated neurons, glial cells, andcells of other lineages.

Gene Expression Analysis

The cells of this invention are also of interest in identifyingexpression patterns of transcripts and newly synthesized proteins thatare characteristic for neural precursor cells, and may assist indirecting the differentiation pathway or facilitating interactionbetween cells. Expression patterns of the differentiated cells areobtained and compared with control cell lines, such as undifferentiatedpPS cells, other types of committed precursor cells (such as pPS cellsdifferentiated towards other lineages, cells committed to the neuronalor glial cell lineage), other types of putative neural stem cells suchas those obtained from neural crest, neurospheres, or spinal chord, orterminally differentiated cells, such as mature neurons, astrocytes,oligodendrocytes, smooth muscle cells, and Schwann cells.

Suitable methods for comparing expression at the protein level includethe immunoassay or immunohistochemistry techniques described above.Suitable methods for comparing expression at the level of transcriptioninclude methods of differential display of mRNA (Liang, Peng, et al.,Cancer Res. 52:6966, 1992), whole-scale sequencing of cDNA libraries,and matrix array expression systems.

The use of microarray in analyzing gene expression is reviewed generallyby Fritz et al Science 288:316, 2000; “Microarray Biochip Technology”, LShi, www.Gene-Chips.com. Systems and reagents for performing microarrayanalysis are available commercially from companies such as Affymetrix,Inc., Santa Clara Calif.; Gene Logic Inc., Columbia Md.; HySeq Inc.,Sunnyvale Calif.; Molecular Dynamics Inc., Sunnyvale Calif.; Nanogen,San Diego Calif.; and Synteni Inc., Fremont Calif. (acquired by IncyteGenomics, Palo Alto Calif.).

Solid-phase arrays are manufactured by attaching the probe at specificsites either by synthesizing the probe at the desired position, or bypresynthesizing the probe fragment and then attaching it to the solidsupport (U.S. Pat. Nos. 5,474,895 and 5,514,785). The probing assay istypically conducted by contacting the array by a fluid potentiallycontaining the nucleotide sequences of interest under suitableconditions for hybridization conditions, and then determining any hybridformed.

An exemplary method is conducted using a Genetic Microsystems arraygenerator, and an Axon GenePix™ Scanner. Microarrays are prepared byfirst amplifying cDNA fragments encoding marker sequences to beanalyzed, and spotted directly onto glass slides To compare mRNApreparations from two cells of interest, one preparation is convertedinto Cy3-labeled cDNA, while the other is converted into Cy5-labeledcDNA. The two cDNA preparations are hybridized simultaneously to themicroarray slide, and then washed to eliminate non-specific binding. Theslide is then scanned at wavelengths appropriate for each of the labels,the resulting fluorescence is quantified, and the results are formattedto give an indication of the relative abundance of mRNA for each markeron the array.

Identifying expression products for use in characterizing and affectingdifferentiated cells of this invention involves analyzing the expressionlevel of RNA, protein, or other gene product in a first cell type, suchas a pluripotent neuronal precursor cell of this invention, or a cellcapable of differentiating along the neuronal or glial pathway; thenanalyzing the expression level of the same product in a control celltype; comparing the relative expression level between the two celltypes, (typically normalized by total protein or RNA in the sample, orin comparison with another gene product expected to be expressed at asimilar level in both cell types, such as a house-keeping gene); andthen identifying products of interest based on the comparativeexpression level.

Drug Screening

Neural precursor cells of this invention can be used to screen forfactors (such as solvents, small molecule drugs, peptides,polynucleotides) or environmental conditions (such as culture conditionsor manipulation) that affect the characteristics of neural precursorcells and their various progeny.

In some applications, pPS cells (undifferentiated or differentiated) areused to screen factors that promote maturation into neural cells, orpromote proliferation and maintenance of such cells in long-termculture. For example, candidate maturation factors or growth factors aretested by adding them to cells in different wells, and then determiningany phenotypic change that results, according to desirable criteria forfurther culture and use of the cells.

Other screening applications of this invention relate to the testing ofpharmaceutical compounds for their effect on neural tissue or nervetransmission. Screening may be done either because the compound isdesigned to have a pharmacological effect on neural cells, or because acompound designed to have effects elsewhere may have unintended sideeffects on the nervous system. The screening can be conducted using anyof the neural precursor cells or terminally differentiated cells of theinvention, such as dopaminergic, serotonergic, cholinergic, sensory, andmotor neurons, oligodendrocytes, and astrocytes.

The reader is referred generally to the standard textbook “In vitroMethods in Pharmaceutical Research”, Academic Press, 1997, and U.S. Pat.No. 5,030,015. Assessment of the activity of candidate pharmaceuticalcompounds generally involves combining the differentiated cells of thisinvention with the candidate compound, either alone or in combinationwith other drugs. The investigator determines any change in themorphology, marker phenotype, or functional activity of the cells thatis attributable to the compound (compared with untreated cells or cellstreated with an inert compound), and then correlates the effect of thecompound with the observed change.

Cytotoxicity can be determined in the first instance by the effect oncell viability, survival, morphology, and the expression of certainmarkers and receptors. Effects of a drug on chromosomal DNA can bedetermined by measuring DNA synthesis or repair. [³H]-thymidine or BrdUincorporation, especially at unscheduled times in the cell cycle, orabove the level required for cell replication, is consistent with a drugeffect. Unwanted effects can also include unusual rates of sisterchromatid exchange, determined by metaphase spread. The reader isreferred to A. Vickers (pp 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

Effect of cell function can be assessed using any standard assay toobserve phenotype or activity of neural cells, such as receptor binding,neurotransmitter synthesis, release or uptake, electrophysiology, andthe growing of neuronal processes or myelin sheaths—either in cellculture or in an appropriate model.

Therapeutic Use

This invention also provides for the use of neural precursor cells torestore a degree of central nervous system (CNS) function to a subjectneeding such therapy, perhaps due to an inborn error in function, theeffect of a disease condition, or the result of an injury.

To determine the suitability of neural precursor cells for therapeuticadministration, the cells can first be tested in a suitable animalmodel. At one level, cells are assessed for their ability to survive andmaintain their phenotype in vivo. Neural precursor cells areadministered to immunodeficient animals (such as nude mice, or animalsrendered immunodeficient chemically or by irradiation) at an observablesite, such as in the cerebral cavity or in the spinal chord. Tissues areharvested after a period of a few days to several weeks or more, andassessed as to whether pPS derived cells are still present.

This can be performed by administering cells that express a detectablelabel (such as green fluorescent protein, or β-galactosidase); that havebeen prelabeled (for example, with BrdU or [³H]thymidine), or bysubsequent detection of a constitutive cell marker (for example, usinghuman-specific antibody). Where neural precursor cells are being testedin a rodent model, the presence and phenotype of the administered cellscan be assessed by immunohistochemistry or ELISA using human-specificantibody, or by RT-PCR analysis using primers and hybridizationconditions that cause amplification to be specific for humanpolynucleotide sequences. Suitable markers for assessing gene expressionat the mRNA or protein level are provided elsewhere in this disclosure.

Various animal models for testing restoration of nervous system functionare described in “CNS Regeneration: Basic Science and ClinicalAdvances”, M. H. Tuszynski & J. H. Kordower, eds., Academic Press, 1999.

Differentiated cells of this invention can also be used for tissuereconstitution or regeneration in a human patient in need thereof. Thecells are administered in a manner that permits them to graft or migrateto the intended tissue site and reconstitute or regenerate thefunctionally deficient area.

Certain neural progenitor cells embodied in this invention are designedfor treatment of acute or chronic damage to the nervous system. Forexample, excitotoxicity has been implicated in a variety of conditionsincluding epilepsy, stroke, ischemia, Huntington's disease, Parkinson'sdisease and Alzheimer's disease. Certain differentiated cells of thisinvention may also be appropriate for treating dysmyelinating disorders,such as Pelizaeus-Merzbacher disease, multiple sclerosis,leukodystrophies, neuritis and neuropathies. Appropriate for thesepurposes are cell cultures enriched in oligodendrocytes oroligodendrocyte precursors to promote remyelination.

By way of illustration, neural stem cells are transplanted directly intoparenchymal or intrathecal sites of the central nervous system,according to the disease being treated. Grafts are done using singlecell suspension or small aggregates at a density of 25,000-500,000 cellsper μL (U.S. Pat. No. 5,968,829). The efficacy of transplants of motorneurons or their precursors can be assessed in a rat model for acutelyinjured spinal cord as described by McDonald et al. (Nat. Med. 5:1410,1999). A successful transplant will show transplant-derived cellspresent in the lesion 2-5 weeks later, differentiated into astrocytes,oligodendrocytes, and/or neurons, and migrating along the cord from thelesioned end, and an improvement in gate, coordination, andweight-bearing.

The neural progenitor cells and terminally differentiated cellsaccording to this invention can be supplied in the form of apharmaceutical composition, comprising an isotonic excipient preparedunder sufficiently sterile conditions for human administration. Forgeneral principles in medicinal formulation, the reader is referred toCell Therapy: Stem Cell Transplantation, Gene Therapy, and CellularImmunotherapy, by G. Morstyn & W. Sheridan eds, Cambridge UniversityPress, 1996; and Hematopoietic Stem Cell Therapy, E. D. Ball, J. Lister& P. Law, Churchill Livingstone, 2000.

The composition may optionally be packaged in a suitable container withwritten instructions for a desired purpose, such as the reconstitutionof CNS function to improve some neurological abnormality.

The following examples are provided as further non-limitingillustrations of particular embodiments of the invention.

EXAMPLES Experimental Procedures

This section provides details of some of the techniques and reagentsused in the Examples below.

hES cells are maintained either on primary mouse embryonic fibroblasts,or in a feeder-free system. The hES cells are seeded as small clusterseither on irradiated mouse embryonic fibroblasts, or on plates coatedwith Matrigel® (1:10 to 1:30 in culture medium). hES cell cultures onfeeder cells are maintained in a medium composed of 80% KO DMEM (Gibco)and 20% Serum Replacement (Gibco), supplemented with 1% non-essentialamino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol and 4 ng/mL humanbFGF (Gibco). Cultures free of feeder cells are maintained in the samemedium that has previously been conditioned by culturing with embryonicfibroblasts, and resupplemented with 4 ng/mL bFGF (replaced daily).

Cells are expanded by serial passaging. The monolayer culture of EScolonies is treated with 1 mg/mL collagenase for 5-20 minutes at 37° C.The cultures are then gently scraped to remove the cells. The clustersare gently dissociated, and replated as small clusters onto fresh feedercells.

Embryoid bodies are produced as follows. Confluent monolayer cultures ofhES cells are harvested by incubating in 1 mg/mL collagenase for 5-20min, following which the cells are scraped from the plate. The cells arethen dissociated into clusters and plated in non-adherent cell cultureplates (Costar) in a medium composed of 80% KO (“knockout”) DMEM (Gibco)and 20% non-heat-inactivated FBS (Hyclone), supplemented with 1%non-essential amino acids, 1 mM glutamine, 0.1 mM β-mercaptoethanol. Thecells are seeded at a 1:1 or 1:2 ratio in 2 mL medium per well (6 wellplate). The EBs are fed every other day by the addition of 2 mL ofmedium per well. When the volume of medium exceeds 4 mL/well, the EBsare collected and resuspended in fresh medium. After 4-8 days insuspension, the EBs are plated onto a substrate and allowed todifferentiate further, in the presence of selected differentiationfactors.

Differentiating into neural precursors is typically performed on wellscoated with fibronectin (Sigma) at a final concentration of 20 μg/mL inPBS. Using 1 mL/well (9.6 cm²), plates are incubated at 4° C. overnightor at room temperature for 4 h. The fibronectin is then removed, and theplates are washed with PBS or KO DMEM once before use.

Immunocytochemistry for NCAM and A2B5 expression is conducted asfollows: Live cells are incubated in primary antibody diluted in culturemedium with 1% goat serum for 15 minutes at 37° C., washed once withmedium, and then incubated with labeled secondary antibody for 15 min.After washing, the cells are then fixed for 15-20 min in 2%paraformaldehyde. For other markers, cultures are fixed for 10-20 minwith 4% paraformaldehyde in PBS, washed 3 times with PBS, permeabilizedfor 2 min in 100% ethanol, and washed with 0.1 M PBS. Cultures are thenincubated in a blocking solution of 0.1 M PBS with 5% NGS (normal goatserum) for at least 1 hour at room temperature. Cultures are thenincubated in primary antibody diluted in 0.1M PBS containing 1% NGS forat least 2 h at room temperature. They are then washed in PBS before a30 min incubation with secondary antibody in the same buffer. Antibodiesused include those shown in Table 1.

TABLE 1 Antibody for Neural Cell Phenotypic Markers Antibody IsotypeWorking Dilution Epitope Specificity Source 5A5 mouse IgM 1:1Polysialylated Developmental studies NCAM hybridoma bank A2B5 mouse IgM1:1 ganglioside ATCC—CRL1520 clone 105 β-tubulin IgG 1:1000 Sigma T-8660GFAP rabbit polyclonal 1:500 DAKO 2-334 IgG GalC mouse IgG3 1:25Boehringer 1351-621

Bead immunosorting is conducted using the following reagents andequipment: magnetic cell separator; Midi MACs™ column; buffer of PBS CMFcontaining 0.5% BSA and 2 mM EDTA; primary antibody against NCAM orA2B5; rat anti-mouse IgG (or IgM) microbeads; pre-separation filter; ratanti-mouse kappa PE; and a FACScan device. Cells are harvested usingtrypsin/EDTA (Gibco) and dissociated. After removing the trypsin, thecells are resuspended in MACs™ buffer. Cells are then labeled withprimary antibody for 6-8 min at room temp., and washed 2 times in MACs™buffer by spinning cells at 300×g for 10 min and aspirating the buffer.The cells are then resuspended in 80 μl (minimum vol.) per 10⁷ cells. 20μl (minimum vol.) MACs Ram™ IgG microbeads per 10⁷ cells are added for15 min at 6-12° C. The sample is then washed 2 times in MACs™ bufferbefore magnetic separation. With the column in the magnetic cellseparator, the cell suspension is applied to the column (LS+ Midi) in˜3-5 mL buffer. Negative cells are passed through by washing 3 timeswith 3 mL of MACs™ buffer. The column is then removed from the magneticfield, and positive cells are eluted with 5 mL of MACs™ buffer.

After separation, A2B5+ or NCAM+ cells are maintained on plates coatedwith poly-lysine and laminin in DMEM/F12 (Biowhittaker) supplementedwith N2 (Gibco 17502-014), B27 (Gibco 17504-010) and the factorsindicated. Source of the factors is shown in Table 2.

TABLE 2 Factors used for Neural Cell Culture Working Growth FactorSource Concentration human EGF R&D Systems 10 ng/mL human bFGF Gibco10-25 ng/mL human CNTF R&D Systems 1-10 ng/mL human PDGF R&D Systems 1ng/mL human IGF-I R&D Systems 1 ng/mL

RT-PCR analysis of expression at the transcription level is conducted asfollows: RNA is extracted from the cells using RNAeasy Kit™ (Qiagen) asper manufacturers instructions. The final product is then digested withDNAse to get rid of contaminating genomic DNA. The RNA is incubated inRNA guard (Pharmacia Upjohn) and DNAse I (Pharmacia Upjohn) in buffercontaining 10 mM Tris ph 7.5, 10 mM MgCl₂, and 5 mM DDT at 37° C. for30-45 min. To remove protein from the sample, phenol chloroformextraction is performed, and the RNA precipitated with 3 M sodiumacetate and 100% cold ethanol. The RNA is washed with 70% ethanol, andthe pellet is air-dried and resuspended in DEPC-treated water.

For the reverse transcriptase (RT) reaction, 500 ng of total RNA iscombined with a final concentration of 1×First Strand Buffer (Gibco), 20mM DDT and 25 μg/mL random hexamers (Pharmacia Upjohn). The RNA isdenatured for 10 min at 70° C., followed by annealing at roomtemperature for 10 min. dNTPs are added at a final concentration of 1 mMalong with 0.5 μL of Superscript 11 RT (Gibco), incubated at 42° C. for50 minutes, and then heat-inactivated at 80° C. for 10 min. Samples arethen stored at −20° C. till they are processed for PCR analysis.Standard polymerase chain reaction (PCR) is performed using primersspecific for the markers of interest in the following reaction mixture:cDNA 1.0 μL, 10×PCR buffer (Gibco) 2.5 μL, 10×MgCl₂ 2.5 μL, 2.5 mM dNTP3.0 μL, 5 μM 3′-primer 1.0 μL, 5 μM 5′-primer, 1.0 μL Taq 0.4 μL,DEPC-water 13.6 pt.

Example 1 NCAM-Positive Cells

This experiment focused on determining whether the human embryonic stemcells (hES) could undergo directed differentiation to NCAM-positiveprogenitor cells. The hES cells were harvested either from mEF-supportedcultures or feeder-free cultures, and then differentiated via embryoidbody (EB) formation in suspension culture using medium containing 20%FBS. The EBs were then plated intact onto fibronectin in DMEM/F12medium, supplemented with N2 supplement (Gibco) and 25 ng/mL human bFGF.After culturing for about 2-3 days, NCAM-positive cells andA2B5-positive cells were identified by immunostaining.

Magnetic bead sorting and immunopanning were both successful inenriching NCAM-positive cells. The starting population of cellstypically contained 25-72% NCAM-positive cells. After immuno-isolation,the NCAM-positive proportion was enriched to 43-72%. Results are shownin Table 3.

TABLE 3 Differentiation and Sorting Conditions for NCAM positive CellsFactors used in Cells staining positively for NCAM hES Cell Line usedDifferentiation Before Positive Negative for Differentiation CultureType of Sort sort sort sort H13 p28 C F N bead sort 33 92 41 H13p28 C FN panning 25 n/a n/a H9 p32 C F N panning 64 72 51 H1 p32 C F N beadsort 27 77 9 H9 p19 C F N bead sort 58 76 32 H9 p31 545.184 C F N beadsort 50 91 67 H1 p40 545.185 C F N bead sort A 65 89 31 H1 p40 545.185 CF N bead sort B 63 81 33 H7NG p28/4 545.187 C F N bead sort A 53 92 45H7NG p28/4 545.187 C F N bead sort A 72 87 50 H1p39 545.189 C F N I Pbead sort 16 43 6 H7 p32 667.004 C F N I P bead sort 25 73 10 H1p43667.010 C F N I P bead sort 47 86 31 H1p44 667.012 C F N I P bead sort52 89 34 H1 p46 667.020 EPFI bead sort 60 23 8 H1 p47 667.031 EPFI—EPFIbead sort 53 91 27 H1 p47 667.033 C F N—F bead sort 41 76 24 H9 p40MG667.038 E P F I bead sort 55 80 25 Factor abbreviations: C—ciliaryneurotrophic factor (CNTF) F—basic fibroblast growth factor (bFGF)N—neurotrophin 3 (NT3) I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF) T—thyroid hormone T₃ Ra—retinoicacid Fk—Forskolin

In the first 10 experiments shown, NCAM positive cells retrieved fromthe sort were plated on poly-L-lysine/laminin in DMEM/F12 with N2 andB27 supplements and 2 mg/mL BSA, 10 ng/mL human CNTF, 10 ng/mL humanbFGF and 1 ng/mL human NT-3. In subsequent experiments, cells weremaintained in DMEM/F12 with N2 and B27 supplements and 10 ng/mL EGF, 10ng/mL bFGF, 1 ng/mL PDGF, and 1 ng/mL IGF-1.

FIG. 1 (Upper Panel) shows the growth curves for the NCAM positivecells. The cells studied in this experiment were prepared by formingembryoid bodies in 20% FBS for 4 days in suspension, then plating onto afibronectin matrix in DMEM/F12 with N2 and B27 supplements and 25 ng/mLbFGF for 2-3 days. The cells were then positively sorted for NCAMexpression, and maintained in a medium containing CNTF, bFGF, and NT3.The sorted cells did not show increased survival relative to theunsorted population. It was found that some of the NCAM positive cellsalso express β-tubulin III, indicating that these cells have thecapacity to form neurons. They also had morphology characteristic ofneuronal cells. There were also A2B5 positive cells within thispopulation, which may represent glial progenitor cells. However, veryfew cells were positive for GFAP, a marker for astrocytes. Although thiscell population proliferated in culture, the proportion of NCAM positivecells (and the capacity to form neurons) diminished after severalpassages.

Example 2 A2B5-Positive Cells

Cells in this experiment were immunoselected for the surface markerA2B5. hES cells were induced to form EBs in 20% FBS. After 4 days insuspension, the EBs were plated onto fibronectin in DMEM/F12 with N2 andB27 supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1 ng/mLhuman IGF-I, and 1 ng/mL human PDGF-AA. After 2-3 days in theseconditions, 25-66% of the cells express A2B5. This population isenriched by magnetic bead sorting to 48-93% purity (Table 4).

TABLE 4 Differentiation and Sorting Conditions for A2B5-positive CellsFactors used in Cells staining positively for NCAM hES Cell Line usedfor Differentiation Positive Negative Differentiation Culture Type ofSort Before sort sort sort H7 p32 667.004 C F N I P bead sort 25 77 10H1p43 667.010 C F N I P bead sort 62 n/a 50 H1 p44 667.012 C F N I Pbead sort 56 89 32 H1 p46 667.020 E P F I bead sort 27 48 2 H1 p47667.032 E P F I bead sort 57 93 30 H9 p40MG 667.038 E P F I bead sort 6693 41 H9 p42 667.041 E P F I bead sort 27 70 6 Factor abbreviations:C—ciliary neurotrophic factor (CNTF) F—basic fibroblast growth factor(bFGF) N—neurotrophin 3 (NT3) I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF) T—thyroid hormone T₃ Ra—retinoicacid Fk—Forskolin

FIG. 2 shows an exemplary procedure for obtaining A2B5-positive cells.Abbreviations used: MEF-CM=medium conditioned by culturing with mouseembryonic fibroblasts; +/−SHH=with or without sonic hedgehog;D/F12=DMEM/F12 medium; N2 and B27, culture supplements (Gibco);EPFI=growth factors EGF, PDGF, bFGF, and IGF-1; PLL=poly-L lysinesubstrate; PLL/FN=substrate of poly-L lysine and fibronectin.

FIG. 1 (Lower Panel) shows the growth curves for the sortedA2B5-positive cells. The cells were maintained in the same mediaformulation on poly-l-lysine coated plates. The cells proliferate whenserially passaged.

Example 3 Maturation of A2B5-Positive Cells

A2B5-positive cells were induced to differentiate by the addition offorskolin. These cells have been assessed through different culturepassages, as shown in Table 5.

TABLE 5 Phenotypic Features of Mature Neural Cells Neuron-like No. ofpassages Method of morphology Cells Staining Positively for: after A2B5sort Maturation visible β-tubulin GFAP GalC A2B5 NCAM 1 PICNT + Fk yes38 ± 9% 13 ± 7% 79 ± 3% 28 ± 6% 4 days 3 PICNT + Fk yes +++ + +++++ ++ 2days 7 +/− EF yes + + ++ +++ − +/− serum Factor abbreviations: C—ciliaryneurotrophic factor (CNTF) F—basic fibroblast growth factor (bFGF)N—neurotrophin 3 (NT3) I—insulin-like growth factor (IGF-1)P—platelet-derived growth factor (PDGF) T—thyroid hormone T₃ Ra—retinoicacid Fk—Forskolin

Even though the cells were sorted for A2B5 expression, the populationdemonstrated the capacity to generate not only oligodendrocytes, andastrocytes, but also a large proportion of neurons. This is surprising:it was previously thought that A2B5 expressing cells were glialprecursors, and would give rise to oligodendrocytes, andastrocytes—while NCAM expressing cells were neuronal precursors, givingrise to mature neurons. This experiment demonstrates that pPS cells canbe differentiated into a cell population that proliferates repeatedly inculture, and is capable of generating neurons and glia.

Example 4 Transplantation of Differentiated Cells into the MammalianBrain

Transplantation of neural precursor cells was done using cells derivedfrom two hES cell lines: the line designated H1, and a geneticallyaltered line designated H7NHG. The H7NHG cell line carries an expressioncassette that permits the cells to constitutively express greenfluorescent protein (GFP).

Neonatal Sprague Dawley rats received unilateral intrastriatal implantsof one of the following cell populations:

-   -   undifferentiated hES cells    -   embryoid bodies derived from hES cells    -   neural precursors sorted for NCAM expression (Example 1)    -   neural precursors sorted for A2B5 expression (Example 2)        Control animals received grafts of irradiated mouse embryonic        fibroblasts upon which the undifferentiated hES cells were        maintained. To determine if cell proliferation occurred after        grafting, some animals were pulsed with intraperitoneal        injections of BrdU, commencing 48 h prior to sacrifice. Fourteen        days after transplantation, the rats were transcardially        perfused with 4% paraformaldehyde and the tissue was processed        for immunohistochemical analysis.

FIG. 3 shows the fluorescence observed in sections from animalsadministered cells expressing GFP. Surviving cells were detected in alltransplanted groups. The undifferentiated cells presented as large cellmasses, suggesting unregulated growth with areas of necrosis andvacuolation of surrounding tissue (Left-side Panels). Immunostaining forAFP in an animal transplanted with HI cells showed that undifferentiatedhES cells transformed into visceral endoderm after transplantation.Embryoid bodies remained in the graft core with little migration, andwere also surrounded by areas of necrosis. (Middle Panels). In contrast,sorted NCAM-positive cells appeared as single cells and showed somedegree of migration distal to the site of implantation.

Example 5 Differentiation to Mature Neurons

To generate terminally differentiated neurons, the first stage ofdifferentiation was induced by forming embryoid bodies in FBS mediumwith or without 10 μM retinoic acid (RA). After 4 days in suspension,embryoid bodies were plated onto fibronectin-coated plates in definedmedium supplemented with 10 ng/mL human EGF, 10 ng/mL human bFGF, 1ng/mL human PDGF-AA, and 1 ng/mL human IGF-1. The embryoid bodiesadhered to the plates, and cells began to migrate onto the plastic,forming a monolayer.

After 3 days, many cells with neuronal morphology were observed. Theneural precursors were identified as cells positive for BrdUincorporation, nestin staining, and the absence of lineage specificdifferentiation markers. Putative neuronal and glial progenitor cellswere identified as positive for polysialylated NCAM and A2B5. Forty oneto sixty percent of the cells expressed NCAM, and 20-66% expressed A2B5,as measured by flow cytometry. A subpopulation of the NCAM-positivecells was found to express β-tubulin III and MAP-2. There was noco-localization with glial markers such as GFAP or GalC. The A2B5positive cells appeared to generate both neurons and glia. Asubpopulation of the A2B5 cells expressed β-tubulin III or MAP-2, and aseparate subpopulation expressed GFAP. Some of the cells with neuronalmorphology double-stained for both A2B5 and NCAM. Both the NCAM positiveand A2B5 positive populations contained far more neurons than glia.

The cell populations were further differentiated by replating the cellsin a medium containing none of the mitogens, but containing 10 ng/mLNeurotrophin-3 (NT-3) and 10 ng/mL brain-derived neurotrophic factor(BDNF). Neurons with extensive processes were seen after about 7 days.Cultures derived from embryoid bodies maintained in retinoic acid (RA)showed more MAP-2 positive cells (˜26%) than those maintained without RA(˜5%). GFAP positive cells were seen in patches. GalC positive cellswere identified, but the cells were large and flat rather than havingcomplex processes.

A summary of cell types and markers expressed at different stages ofdifferentiation is provided in Table 6.

TABLE 6 Phenotypic Markers (Immunocytochemistry) Undifferentiated hEScolonies NCAM-positive progenitors A2B5 positive progenitors Tra-1-60 +Nestin subset Nestin subset Tra-1-81 + A2B5 subset NCAM subset SSEA-4 +β-tubulin III subset β-tubulin III subset β-tubulin III + + Map-2 subsetMap-2 subset Nestin − GFAP − GFAP rare Map-2 − GalC − GalC −Neurofilament (NF) − AFP − AFP − GFAP − muscle-specific actin −muscle-specific actin − GalC − α-fetoprotein − muscle-specific actin −NCAM − A2B5 − Neurons Astrocytes Oligodendrocytes β-tubulin III + GFAP +GaIC + MAP-2 + Neurofilament (NF) subset GABA subset tyrosinehydroxylase subset glutamate subset glycine subset

The presence of neurotransmitters was also assessed. GABA-immunoreactivecells were identified that co-expressed β-tubulin III or MAP2, and hadmorphology characteristic of neuronal cells. Occasional GABA-positivecells were identified that did not co-express neuronal markers, but hadan astrocyte-like morphology. Neuronal cells were identified thatexpressed both tyrosine hydroxylase (TH) and MAP-2. Synapse formationwas identified by staining with synaptophysin antibody.

FIG. 4 shows TH staining in cultures differentiated from the H9 line ofhuman ES cells. Embryoid bodies were maintained in 10 μM retinoic acidfor 4 days, then plated onto fibronectin coated plates in EGF, basicFGF, PDGF and IGF for 3 days. They were next passaged onto laminin in N2medium supplemented with 10 ng/mL NT-3 and 10 ng/mL BDNF, and allowed todifferentiate further for 14 days. The differentiated cells were fixedwith 2% formaldehyde for 20 min at room temp, and then developed usingantibody to TH, a marker for dopaminergic cells.

Example 6 Calcium Imaging

Standard fura-2 imaging of calcium flux was used to investigate thefunctional properties of the hES cell derived neurons. Neurotransmittersstudied included GABA, glutamate (E), glycine (G), elevated potassium(50 mM K⁺ instead of 5 mM K⁺), ascorbic acid (control), dopamine,acetylcholine (ACh) and norepinephrine. The solutions contained 0.5 mMof the neurotransmitter (except ATP at 10 NM) in rat Ringers (RR)solution: 140 mM NaCl, 3 mM KCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM HEPESbuffer, and 10 mM glucose. External solutions were set to pH 7.4 usingNaOH. Cells were perfused in the recording chamber at 1.2-1.8 mL/min,and solutions were applied by bath application using a 0.2 mL loopinjector located ˜0.2 mL upstream of the bath import. Transient rises incalcium were considered to be a response if the calcium levels roseabove 10% of the baseline value within 60 sec of application, andreturned to baseline within 1-2 min.

FIG. 5 shows the response of neural-restricted precursors to variousneurotransmitters. Panel A shows the ratio of emission data from singlecells on two different coverslips. Addition of the neurotransmitters isindicated above by labeled triangles.

Panel B shows the frequency of cells tested that responded to specificneurotransmitters. Panel C shows the combinations of neurotransmitterresponses observed. Of the 53 cells tested, 26 responded to GABA,acetylcholine, ATP and elevated potassium. Smaller subsets of thepopulation responded to other combinations of agonists. Only 2 of thecells failed to respond to any of the agonists applied.

Example 7 Electrophysiology

Standard whole-cell patch-clamp technique was conducted on the hES cellderived neurons, to record ionic currents generated in voltage-clampmode and the action potential generated in current-clamp mode. Theexternal bath solution was rat Ringers solution (Example 6). Theinternal solution was 75 mM potassium-aspartate, 50 mM KF, 15 mM NaCl,11 mM EGTA, and 10 mM HEPES buffer, set to pH 7.2 using KOH.

All 6 cells tested expressed sodium and potassium currents, and firedaction potentials. Passive membrane properties were determined withvoltage steps from −70 to −80 mV; and produced the following data:average capacitance (C_(m))=8.97±1.17 pF; membrane resistance(R_(m))=487.8±42.0 MΩ; access resistance (R_(a))=23.4±3.62 Ma Ioniccurrents were determined by holding the cells at −100 mV, and steppingto test voltages between −80 and 80 mV in 10 mV increments, producingthe following data: average sodium current I_(Na)=−531.8±136.4 pA;average potassium current I_(K)=441.7±113.1 pA;I_(Na)(density)=−57.7±7.78 pA/pF; I_(K)(density)=48.2±10.4 pA/pF.

FIG. 6 shows results from a typical experiment. Panel A shows sodium andpotassium currents observed in two cells depolarized to test potentialsbetween −80 and 80 mV from a holding potential of −100 mV. Panel B showsthe inward (Nat) and outward (K⁺) peak current-voltage relationshipsobserved. Sodium current activates between −30 and 0 mV, reaching a peakat −10 or 0 mV. Potassium current activates above −10 mV, becoming equalor larger in magnitude than the sodium current at voltages between 20and 40 mV. Panel C shows action potentials generated by the same cells nresponse to depolarizing stimuli. Cell membranes were held at voltagesbetween −60 and −100 mV in −80 or −150 pA of current, and depolarizedfor short durations

Example 8 Dopaminergic Cells Derived from Neural Progenitor Cells

Embryoid bodies were cultured in suspension with 10 μM retinoic acid for4 days, then plated into defined medium supplemented with EGF, bFGF,PDGF, and IGF-1 for 3-4 days. Cells were then separated by magnetic beadsorting or immunopanning into A2B5-positive or NCAM-positive enrichedpopulations.

The immuno-selected cells were maintained in defined medium supplementedwith 10 ng/mL NT-3 and 10 ng/mL BDNF. After 14 days, 25±4% of theNCAM-sorted cells were MAP-2 positive—of which 1.9±0.8% wereGABA-positive, and 3±1% were positive for tyrosine hydroxylase (TH): therate-limiting enzyme for dopamine synthesis, generally considered to berepresentative of dopamine-synthesizing cells.

In the cell population sorted for NCAM, the cells that were NCAM+ve didnot express glial markers, such as GFAP or GalC. These data indicatethat a population comprising neuron restricted precursors can beisolated directly from hES cell cultures, essentially uncontaminatedwith glial precursors.

Cells sorted for A2B5, on the other hand, have the capacity to generateboth neurons and astrocytes. After the enrichment, the cells were placedinto defined media supplemented with NT-3 and BDNF and allowed todifferentiate for 14 days. Within the first 1-2 days after plating,cells in the A2B5 enriched population began to extend processes. Aftertwo weeks, cells took on the morphology of mature neurons, and 32±3% ofthe cells were MAP-2 positive. Importantly, 3±1% of the MAP-2 cells wereTH-positive, while only 0.6±0.3% were GABA immunoreactive. These dataindicate that a population of cells can be obtained from hES cells thatcomprise progenitors for both astrocytes and neurons, including thosethat synthesize dopamine.

Further elaboration of conditions for obtaining TH-expression neuronswas conduced as follows. Embryoid bodies were generated from confluenthES cells of the H7 line at passage 32 by incubating in 1 mg/mLcollagenase (37° C., 5-20 min), scraping the dish, and placing the cellsinto non-adherent culture plates (Costar®). The resulting EBs werecultured in suspension in media containing FBS and 10 μM all-transretinoic acid. After four days, the aggregates were collected andallowed to settle in a centrifuge tube. The supernatant was thenaspirated, and the aggregates were plated onto poly L-lysine andfibronectin coated plates in proliferation medium (DMEM/F12 1:1supplemented with N2, half-strength B27, 10 ng/mL EGF (R & D Systems),10 ng/mL bFGF (Gibco), 1 ng/mL PDGF-AAA (R & D Systems), and 1 ng/mLIGF-1 (R & D Systems).

The EBs were allowed to attach and proliferate for three days; thencollected by trypsinizing ˜1 min (Sigma) and plated at 1.5×10⁵cells/well onto poly 1-lysine and laminin coated 4-well chamber slidesin proliferation medium for one day. The medium was then changed toNeural Basal medium supplemented with B27, and one of the followinggrowth cocktails:

-   -   10 ng/mL bFGF (Gibco), 10 ng/mL BDNF, and 10 ng/mL NT-3    -   10 ng/mL bFGF, 5000 ng/mL sonic hedgehog, and 100 ng/mL FGF8b    -   10 ng/mL bFGF alone        The cells were maintained in these conditions for 6 days, with        feeding every other day. On day 7, the medium was changed to        Neural Basal medium with B27, supplemented with one of the        following cocktails:    -   10 ng/mL BDNF, 10 ng/mL NT-3    -   1 μM cAMP, 200 μM ascorbic acid    -   1 μM cAMP, 200 μM ascorbic acid, 10 ng/mL BDNF, 10 ng/mL NT-3        The cultures were fed every other day until day 12 when they        were fixed and labeled with anti-TH or MAP-2 for        immunocytochemistry. Expression of the markers was quantified by        counting four fields in each of three wells using a 40×        objective lens.

Results are shown in Table 7. Initial culturing in bFGF, BDNF and NT-3yielded the highest proportion of TH positive cells.

TABLE 7 Conditions for Producing Dopaminergic Neurons Culture conditions% MAP-2 cells that are days 1-6 days 6-12 % MAP-2 positive TH positiveB, N, F B, N 26% 5.5% B, N, F CA, AA 35% 4.0% B, N, F CA, AA, B, N 25%8.7% F, F8, S B, N 37% 3.7% F, F8, S CA, AA 34% 3.9% F, F8, S CA, AA, B,N 21% 5.8% F B, N 28% 3.5% F CA, AA 26% 4.1% F CA, AA, B, N 22% 5.7%Factor abbreviations: F—basic fibroblast growth factor (bFGF) F—FGF8N—neurotrophin 3 (NT3) B—brain-derived neurotrophic factor (BDNF)S—sonic hedgehog CA—cAMP AA—ascorbic acid

It is understood that certain adaptations of the invention described inthis disclosure are a matter of routine optimization for those skilledin the art, and can be implemented without departing from the spirit ofthe invention, or the scope of the appended claims.

We claim: 1-30. (canceled)
 31. A first and second cell populationcomprising a) a first in vitro population of cells comprising cellsexpressing stage specific embryonic antigen 3 (SSEA 3) and stagespecific embryonic antigen 4 (SSEA 4) and markers detectable usingantibodies TRA-1-60 and TRA-1-81; and b) a second in vitro cellpopulation comprising progeny of a portion of the first population ofcells, wherein the progeny express NCAM and is a neural precursor. 32.The first and second cell populations of claim 31, wherein the first andsecond populations of cells are contained in separate containers.
 33. Afirst and second cell population comprising a) a first in vitropopulation of cells comprising cells expressing stage specific embryonicantigen 3 (SSEA 3) and stage specific embryonic antigen 4 (SSEA 4) andmarkers detectable using antibodies TRA-1-60 and TRA-1-81; and b) asecond in vitro cell population comprising progeny of a portion of thefirst population of cells, wherein the progeny express A2B5 and aremultipotent neural progenitor cells.
 34. The first and second cellpopulations of claim 33, wherein the first and second populations ofcells are contained in separate containers.
 35. The first and secondcell populations of claim 33, wherein the second population of cellsexpress NCAM.
 36. The first and second cell populations of claim 33,wherein the second population of cells express .beta.-tubullin III. 37.The first and second cell populations of claim 33, wherein the secondpopulation of cells express Map-2.
 38. The first and second cellpopulations of claim 33, wherein the second population of cells expressGFAP.
 39. A first and second cell population comprising a) a first invitro population of cells comprising cells expressing stage specificembryonic antigen 3 (SSEA 3) and stage specific embryonic antigen 4(SSEA 4) and markers detectable using antibodies TRA-1-60 and TRA-1-81;and b) a second in vitro cell population comprising progeny of a portionof the first population of cells, wherein the progeny express tyrosinehydroxylase and wherein the second population of cells express Map-2.